Immobilized carbohydrate biosensor

ABSTRACT

A biosensor in which a carbohydrate or a derivative of a carbohydrate is used to generate a detectable signal by way of the specific binding to a protein, a virus or a cell.

REFERENCE TO A RELATED APPLICATION

This is a continuation application of U.S. patent application Ser. No.09/766,659 filed Jan. 23, 2001 now U.S. Pat. No. 6,887,689 which in turnis a continuation of U.S. patent application Ser. No. 08/356,229 filedDec. 19, 1994, now U.S. Pat. No. 6,231,733 which in turn is acontinuation of PCT/SE94/00343 filed Apr. 18, 1994 which claims thebenefit of Swedish priority document 9301270-6 filed Apr. 19, 1993, allof which are relied on and incorporated herein by reference.

The present invention relates to a biosensor in which a carbohydrate ora derivative thereof is used to generate a detectable signal via thespecific binding of a protein, a virus or a cell.

BACKGROUND

Biosensors are characterised by a physical or chemical signaltransducer, which response is activated by a specific interactionbetween a biochemical structure (which directly or indirectly has beenbound to the transducer) and one or several analytes.

Biosensors are used to detect the analyte/analytes and in certain casesalso for quantification of the analyte/analytes.

The advantages of the biosensor are that a physical or chemicaltransducer has been made specific so that a general physical or chemicalparameter (e.g. temperature, pH, optical density) can be used for thedetection of one specific substance in a complex mixture of non-specificsubstances.

The limitations of the biosensor are the specificity of the biochemicalstructure bound to the transducer, the range of specificity andstability, and, that the transducer signal has to be made independent ofthe background changes in the parameter that the transducer ismeasuring. In Methods of Enzymology, volume 137, several articles aredescribing different aspects of biosensors.

DEFINITIONS

Biosensor—physical or chemical signal transducer, e.g. photometer,chemical electrode, temperature or pressure signal transducer, whichdirectly or indirectly has been connected with a biochemical structure.In previous biosensors one has preferentially used an enzyme, a specificprotein or antibody as the biochemical structure and in this way thebiosensors have been given the property of being able to detectsubstances which specifically bind to the biochemical structure in aqualitative or quantitative way.

Reflection measurement—measurement of the intensity of light reflectedfrom a surface where the properties of the surface influences thereflection, e.g. biomolecules which change the refraction index of thesurface.

Polarisation measurement—measurement of the polarisation of polarisedlight, usually as the angle of polarisation, which is depending on thebinding of biomolecules, virus or cells.

Surface plasmon spectroscopy—optical physical measurement techniquewhich utilise the surface plasmon condition of thin metal surfaces,which can be used to measure small changes of refraction index with highsensitivity, e.g. as caused by the presence of biomolecules on thesurface.

Ellipsometry—optical physical measurement technique which can be used tomeasure small changes of refraction index at surfaces with highsensitivity, by measuring changes in elliptisity of polarised light,e.g. as caused by the presence of biomolecules on the surface.

Piezoelectric crystal—crystal which frequency can be influenced bychanges of mass or pressure which can be measured electrically, forexample the change of mass caused by the presence of biomolecule(s),virus or cell(s) bound to the crystal surface.

Electrochemical electrode—measuring device which generates an electricalsignal caused by an electrochemical reaction at the electrode which isrelated to a chemical parameter, e.g. pH, PO₂, pCO₂, the values of whichcan vary because of the presence of analyte(s) in a sample specific fora compound bound to the measuring device.

Thermistor—electrical resistance device which changes resistance withthe temperature; biochemical reactions are characterised by e.g.specific values of heat consumption/formation, which can be registeredvia the thermistor.

A large amount of the carbohydrate sequences present in glycoproteins orin glycolipids, and usually also smaller fragments of these sequences,have shown biospecific binding to proteins, virus or cells.

The present invention describes a biosensor where this specificity isused for determination of such a component in a sample. The invention ischaracterised by that the carbohydrate or a derivative thereof is boundto a surface in the biosensor.

As carbohydrate, one can use fragments (oligosaccharides) of thecarbohydrate sequences found in glycoproteins or in glycolipids and onecan also use smaller fragments of these sequences, i.e. disaccharide,trisaccharide, tetrasaccharide or a pentasaccharide, because this sizeusually is sufficient for the oligosaccharide to bind a protein, virusor a cell in a biospecific manner. A review or such carbohydratesequences can be found in e.g. Chemistry and Physics of Lipids, vol. 42,p. 153–172, 1986, and in Ann. Rev. Biochem., vol. 58, p. 309–350.

The oligosaccharide is usually modified in the reducing end with anaglycon, which is composed of a glycosidically bound organic group whichis suitable for binding to the surface in the biosensor. Examples ofaglycons are OEtSEtCONHNH₂, —OEtSPhNH₂, etc. The binding to the surfacein the biosensor can be done directly or via proteins, e.g. bovine serumalbumine or via a chemical structure which has been adsorbed or whichhas been covalently bound to the surface. Such a chemical structure cancontain reactive organic groups such as carboxyl-, sulfonate, cyanate,epoxy-, aldehyde groups or other groups suitable for chemicalconjugation with for example an amine or thiol group in the aglycon.

More specific examples of analytes which can be analysed with biosensoraccording to the present invention are lectins, antibodies againstcarbohydrates, pathogenic virus or bacteria, such as urinary tractbacteria (e.g. P-fimbriated E. coli) or pathogens of the respiratorytract, and bacteria which cause infections/diarrhea in gastrointestinaltract.

Non-limiting examples of carbohydrate structures of interest and whichcan be used in the form of a carbohydrate derivative in a biosensoraccording to the invention, are monosaccharides, disaccharides,trisaccharides and higher oligosaccharides which show biologicalactivity or which has the ability to specifically bind one or morebiomolecules or a group of biomolecules. Examples of biomolecules areother saccharides, peptides and proteins. Examples of such carbohydratesequences are the blood group determinants (for example A, B, H,Lewis-a, Lewis-b, Lewis-x, Lewis-y), cancer-associated carbohydratesequences, carbohydrate sequences (often di, tri- or tetrasaccharides)which bind to pathogenic bacteria/toxins or virus of for example therespiratory, the gastro-intestinal or the urinary tract, carbohydratesequences which bind to proteins/cells/white blood cells associated withinflammatory reactions (for example selectin-carbohydrate reactions).

These and other carbohydrate structures which can be used in a biosensoraccording to the present invention often contain one or more of thefollowing monosaccharides (or a derivative or an analog of any of these)which are (∝ or β-glycosidically bound: hexosamine, fucose, mannose,glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, xylose,galactose, or another monosaccharide. These components are usuallypresent in for example pyranose or furanose form.

Examples of carbohydrate derivatives are derivatives where thecarbohydrate or a derivative or an analog, are modified in the reducingend with an 0-, N-, C- or S-glycosidically bound aglycon which can be analiphatic or an aromatic compound, an amino acid-, peptide- or proteinmolecule or a derivative thereof. The aglycon part can thus be composedof for example an 0-, N-, C- or S-glycosidically bound aliphatic oraromatic compound which is bound to an amino acid-, peptide- or proteinmolecule or a derivative thereof. Examples of carbohydrate derivativeswhich can be used according to the invention are structures in which oneor more of the hydroxyl groups in the carbohydrate, in addition to orinstead of the hydroxyl group in the reducing end of the carbohydratepart, have been modified with an organic or inorganic group. This can beof interest, for example to increase/modify the biological activity orto facilitate the binding to the biosensor surface according to theinvention.

The aglycon part or another group can be used for adsorption or covalentbinding of the carbohydrate derivative to the surface of the biosensorand can be used in the invention as a spacer molecule between thebiosensor surface and the carbohydrate part to minimise stericalhindrance in the binding of the analyte to the carbohydrate part in thebiosensor according to the invention.

The aliphatic or aromatic compound in the aglycon can for exampleconsist of structures of the type —R—X, where R— consists of an organiccompound, for example an alkyl chain of the type (—CH₂)_(n), in which nis an integer, e.g. in the interval 2 to 8, or is composed of anaromatic group-containing structure, and where —X is for example astructure of the type —S—, amide (—NH—CO— or CO—NH—), amine (—NH—), a—N═N— group or another group suitable for binding to the surface in thebiosensor or to a protein (i.e. in the latter case the carbohydratederivative is a neoglycoprotein). When the carbohydrate derivative is aneoglycoprotein R can be used as a spacer between the protein part andthe carbohydrate part. The spacer often has a functional part (—X—above) which has been used in the binding to the protein.

Suitable spacer and functional group is chosen by the person skilled inthe art and does not limit the scope of the invention.

The carbohydrate derivative can also, according to the invention, becomposed of a natural, in vitro isolated glycoprotein or a recombinantglycoprotein or a glycopeptide. This type of derivative can be adsorbedto the surface in the biosensor, for example a gold- or silica surfaceor another surface which adsorbs proteins, lipids or peptides.

In the case a covalent binding is desired one can, as in the case whenthe carbohydrate derivative is a neoglycoprotein, use for example theprotein part's amino-, carboxyl-, or thiol groups for binding to thesurface in the biosensor. This (as for the synthesis of theneoglycoprotein from carbohydrate spacer and protein) can be done withthe standard techniques which normally are used for modification ofproteins and for immobilisation of proteins to solid supports (see forexample methods mentioned in Methods of Enzymology, volumes 44, 102, and135), and the choice of suitable technology is made by the personskilled in the art in every specific case. Examples of methods arecoupling or activation of carboxyl groups with carbodiimide reagents,N-hydroxysuccinimide reagents, of hydroxyl groups with CNBr, sulphonylchloride (tresyl chloride, tosyl chloride), divinyl sulphone, periodate(gives aldehyde groups), thiol groups are activated with thiol reagentsof the type N-succinionidyl 3-(2-pyridyldithio)propionate, etc.

As examples of surfaces according to the invention may be mentioned:

Carbohydrate-R—X-Biosensor surface or Carbohydrate-R—X-Protein-Biosensorsurface,

where Carbohydrate, R and X have been exemplified above. X and Proteincan be directly adsorbed on the Biosensor surface above, but between Xand Biosensor surface above and between Protein and Biosensor surfaceabove can also a chemical group be present, for example a —CO—CH₂CH₂—S—group, i.e. for example:

Carbohydrate-R—NH—CO—CH₂—CH₂—S-Biosensor surface or

Carbohydrate-R—X-Protein-NH—CO—CH₂—CH₂—S-Biosensor surface.

As protein one can use for example bovine serum albumin, but all for theapplication suitable types of proteins can be used in the carbohydratederivative-based biosensor according to the invention.

The biosensor according to the invention can be designed in a variety ofconfigurations. Examples are:

a) planar carbohydrate surface which easily can be contacted with thesample, for example a surface designed as a dipstick, this surface canbe placed in a measuring device for optical reflectance measurement inair.

b) Flow system with flow cell, the surface of which is modified withcarbohydrate and where the signal is transferred with optical,electrochemical, thermical or gravimetric method and where the measuringdevice is placed in, or in close connection with the cell.

c) Cuvette or other sample cell, which has been connected with a signaltransducer equipped with carbohydrate to which the sample is added.

d) Planar carbohydrate surface which consists of part of the signaltransducer which with ease can be brought into contact with the samplefor a suitable time, whereafter the sample is removed and the surface ofthe signal transducer is characterised with a physical measuring method,for example electronic measurement, gravimetric measurement or thermalmeasurement.

In some situations, e.g. to increase the biosensor signal in themeasurement of low concentrations of cells, it can be advantageous inthe measurement of the analyte with the biosensor to add, after thebinding of the analyte to the carbohydrate surface, micro particlesmodified with carbohydrate specific for the bound cell.

The surface of the biosensor can be, for example a gold surface or amodified gold surface, a plastic surface which has been modified with agold surface, silver surface or another metallic surface, ormodifications thereof with polymers to which chemical coupling ofcarbohydrate can be carried out.

Below are given non-limiting examples of carbohydrate surfaces which canbe used in biosensors according to the invention for binding andanalysis/determination of pathogenic bacteria of the urinary tract.

EXAMPLE

One example was performed as follows: Silica surface coated with a goldlayer was modified with mercaptopropionic acid by dipping the surface ina 5 mM solution of the acid. The carboxyl groups were modified withcarbodiimide (EDC) for 2 hours, whereafter digalactoside with aglycon(Gal _(∝) 1-4Galβ-OEtSEtCONHNH₂), was coupled to the EDC-activatedsurface for 12 hours at pH 8.5 and the surface was then rinsed withbuffer.

The thus obtained gold surface modified with digalactoside was dippedfor 60 minutes (this time can be varied) in a sample with bacteria ofthe urinary tract (P-fimbriated E. coli) containing Gal ∝1-4Gal-specific receptor protein, followed by rinsing of the surfacewith distilled water for 2 minutes. Another gold surface modified in thesame way with Gal ∝ 1-4Gal, was dipped in a sample containing anothernon-infectious E. coli strain which lack the Gal ∝ 1-4Gal-specificreceptor protein. The extent of binding of the different bacteria to thesurfaces was compared with electrom microscopy. The bacteria with theGal ∝ 1-4Gal-receptor bound to the surface to a ca 10–15 times higherextent than the other bacteria. The binding of P-fimbriated E. coli to agold surface modified with mercapto-propionic acid alone, was ca 20times lower than to the Gal ∝ 1-4Gal-modified surface.

Alternative non-limiting examples are given below in which aneoglycoprotein was bound covalently or adsorbed diredly on a surfacefor use in biosensor according to the invention.

In procedure B, Gal ∝ 1-4GalβOCH₂CH₂SCH₂CH₂C(O)—NHNH-BSA was coupled tothe same type of EDC-activated gold plate as in the procedure above. TheGalabiose-BSA derivative (0.1 mg/ml) was dissolved in 0.1 M boronate, pH8.5 and EDC-activated plates were immersed in this solution for 1 hour.Subsequently the plates were immersed in a BSA solution (3 mg/ml) inphosphate buffer for 1 minute and rinsed with buffer and distilled waterand stored as above.

In procedure C, Gold plates (not pretreated with mercaptopropionic acid)were immersed in a solution of Gal ∝ 1-4Galβ-BSA (0.1 mg/ml) in 0.1 Msodium phosphate, pH 6.0, for 1 hour and subsequently immersed in theabove BSA solution (3 mg/ml) for 1 minute, rinsed with buffer anddistilled water and stored as above.

These latter biosensor surfaces showed similar characteristics and lowback-ground binding of bacteria as surface in the first example above.

1. An immobilized carbohydrate derivative biosensor, comprising: asurface, a carbohydrate derivative which is capable of binding to atleast one member selected from the group consisting of a protein, avirus and a cell, wherein the carbohydrate derivative, containing atleast one O-, N-, C- or S-glycosidically bound aglycon, in which theaglycon contains at least one aliphatic or aromatic compound, and thealiphatic or aromatic compound consists of a spacer and a binding group,the binding group comprises a member selected from the group consistingof —S, —NH—CO—, —CO—NH—, —NH—, and —N═N—, and the binding group is boundto the surface.
 2. The biosensor according to claim 1, wherein saidcarbohydrate derivative is a fragment of a naturally occurringcarbohydrate sequence.
 3. The biosensor according to claim 2, whereinthe fragment of a naturally occurring carbohydrate sequence is a memberselected from the group consisting of a mono-, di-, tri-, tetra-, orpenta-saccharide sequence.
 4. The biosensor according to claim 2,wherein the fragment of a naturally occurring carbohydrate sequence isbindable to P-fimbriated E. coli.
 5. The biosensor according to claim 1,wherein said binding group is chemically bound or is bound viaadsorption to the surface of the biosensor.
 6. The biosensor accordingto claim 1, wherein said surface comprises a signal transducer.
 7. Thebiosensor according to claim 6, wherein said signal transducer is achemical transducer.
 8. The biosensor according to claim 6, wherein saidsignal transducer is a physical transducer.
 9. The biosensor accordingto claim 1, wherein said surface comprises a means for monitoring aphysical signal.
 10. The biosensor according to claim 9, wherein saidmeans for monitoring a physical signal is at least one member selectedfrom the group consisting of a photometer, a chemical electrode, anelectrochemical electrode, a temperature signal transducer, and apressure signal transducer.
 11. The biosensor according to claim 1,wherein said carbohydrate derivative comprises at least one componentselected from the group consisting of hexosamine-, fucose-, galactose-,glucose-, mannose-, xylose-, a N-acetylneuraminic acid residue, andanalogs thereof.
 12. The biosensor according to claim 11, wherein thecarbohydrate derivative has been derivatized in at least one hydroxylgroup or amino group thereof with an organic or inorganic group.
 13. Thebiosensor according to claim 1, in which the aglycon part of thecarbohydrate derivative contains an amino acid, peptide, or proteinmolecule.
 14. The biosensor according to claim 1, in which thecarbohydrate derivative comprises at least one of a glycoprotein and aneoglycoprotein.
 15. The biosensor according to claim 1, wherein saidsurface is associated with an optical sensor which gives a signal changeupon binding of a protein, a virus or a cell to the carbohydratederivative bound via the spacer to the surface.
 16. The biosensoraccording to claim 15, wherein the optical sensor functions by at leastone method selected from the group consisting of surface plasmonchanges, ellipsometry, reflection measurement and polarizationmeasurement.
 17. The biosensor according to claim 1, in which thesurface is associated with a member selected from the group consistingof a piezoelectric crystal, an electrochemical electrode and athermistor.
 18. The biosensor according to claim 1, wherein said surfaceof the biosensor comprises gold.
 19. A method of using the biosensoraccording to claim 1 to determine the presence or amount of a protein, avirus or a cell, comprising the steps of: exposing the biosensor to asample containing a protein, a virus or a cell to be measured, binding aprotein, virus or cell to the biosensor, and measuring the presence oramount of the protein, virus or cell in the sample.
 20. An immobilizedcarbohydrate derivative biosensor, comprising: a surface, a bindinggroup, a carbohydrate derivative which is capable of binding to at leastone member selected from the group consisting of a protein, a virus anda cell, wherein the carbohydrate derivative, containing at least one O-,N-, C- or S-glycosidically bound aglycon, in which the aglycon containsat least one aliphatic or aromatic compound, and the carbohydratederivative contains a binding group, a chemical group is present betweenthe surface and the binding group, wherein the chemical group is a—CO—CH₂CH₂S— group, and the chemical group is bound to the surface. 21.The biosensor according to claim 20, wherein said carbohydratederivative is a fragment of a naturally occurring carbohydrate sequence.22. The biosensor according to claim 21, wherein the fragment of anaturally occurring carbohydrate sequence is selectively bindable to atleast one member selected from the group consisting of a lectin, acancer cell, a protein associated with a blood group determinant, apathogenic bacteria, a pathogenic virus, a pathogenic toxin, a proteinassociated with an inflammatory reaction, and a cell associated with aninflammatory reaction.
 23. The biosensor according to claim 21, whereinthe fragment of a naturally occurring carbohydrate sequence is a memberselected from the group consisting of a mono-, di tri-, tetra-, orpenta-saccharide sequence.
 24. The biosensor according to claim 20,wherein said surface comprises a signal transducer.
 25. The biosensoraccording to claim 20, wherein said surface comprises a means formonitoring a physical signal.
 26. The biosensor according to claim 25,wherein said means for monitoring a physical signal is at least onemember selected from the group consisting of a photometer, a chemicalelectrode, an electrochemical electrode, a temperature signaltransducer, and a pressure signal transducer.
 27. The biosensoraccording to claim 20, wherein said surface is associated with anoptical sensor which gives a signal change upon binding of a protein, avirus or a cell to the carbohydrate derivative.
 28. The biosensoraccording to claim 27, wherein the optical sensor functions by at leastone method selected from the group consisting of surface plasmonchanges, ellipsometry, reflection measurement and polarizationmeasurement.
 29. The biosensor according to claim 20, in which thesurface is associated with a member selected from the group consistingof a piezoelectric crystal, an electrochemical electrode and athermistor.
 30. The biosensor according to claim 20, wherein saidsurface of the biosensor comprises gold.
 31. A method of using thebiosensor according to claim 20 to determine the presence or amount of aprotein, a virus or a cell, comprising the steps of: exposing thebiosensor to a sample containing a protein, a virus or a cell to bemeasured, binding a protein, virus or cell to the biosensor, andmeasuring the presence or amount of the protein, virus or cell in thesample.
 32. The biosensor according to claim 20, further comprising a:spacer molecule which comprises an alkyl chain of the structure(—CH₂)_(n), in which n is an integer from 2 to
 8. 33. An immobilizedcarbohydrate derivative biosensor, comprising: a surface, a carbohydratederivative which is capable of binding to at least one member selectedfrom the group consisting of a protein, a virus and a cell, wherein thecarbohydrate derivative, containing at least one O-, N-, C- orS-glycosidically bound aglycon, in which the aglycon contains at leastone aliphatic or aromatic compound, and the carbohydrate derivative isbound to a spacer molecule containing a binding group, a chemical groupis present between the surface and the binding group, a protein islinked between the spacer and the binding group, wherein the chemicalgroup is a —CO—CH₂CH₂S— group, and the chemical group is bound to thesurface.
 34. The biosensor according to claim 33, wherein the proteincomprises bovine serum albumin.
 35. The biosensor according to claim 33,wherein said carbohydrate derivative is a fragment of a naturallyoccurring carbohydrate sequence, which fragment is bindable to at leastone member selected from the group consisting of a protein, a virus anda cell.
 36. The biosensor according to claim 35, wherein the fragment ofa naturally occurring carbohydrate sequence is a member selected fromthe group consisting of a mono-, di-, tri-, tetra-, or penta-saccharidesequence.
 37. The biosensor according to claim 35, wherein the fragmentof a naturally occurring carbohydrate sequence is selectively bindableto at least one member selected from the group consisting of a lectin, acancer cell, a protein associated with a blood group determinant, apathogenic bacteria, a pathogenic virus, a pathogenic toxin, a proteinassociated with an inflammatory reaction, and a cell associated with aninflammatory reaction.
 38. The biosensor according to claim 33, whereinsaid surface comprises a signal transducer.
 39. The biosensor accordingto claim 33, wherein said surface comprises a means for monitoring aphysical signal.
 40. The biosensor according to claim 39, wherein saidmeans for monitoring a physical signal is at least one member selectedfrom the group consisting of a photometer, a chemical electrode, anelectrochemical electrode, a temperature signal transducer, and apressure signal transducer.
 41. The biosensor according to claim 33,wherein said surface is associated with an optical sensor which gives asignal change upon binding of a protein, a virus or a cell to thecarbohydrate derivative.
 42. The biosensor according to claim 41,wherein the optical sensor functions by at least one method selectedfrom the group consisting of surface plasmon changes, ellipsometry,reflection measurement and polarization measurement.
 43. The biosensoraccording to claim 33, in which the surface is associated with a memberselected from the group consisting of a piezoelectric crystal, anelectrochemical electrode and a thermistor.
 44. The biosensor accordingto claim 33, wherein said surface of the biosensor comprises gold.
 45. Amethod of using the biosensor according to claim 33 to determine thepresence or amount of a protein, a virus or a cell, comprising the stepsof: exposing the biosensor to a sample containing a protein, a virus ora cell to be measured, binding a protein, virus or cell to thebiosensor, and measuring the presence or amount of the protein, virus orcell in the sample.
 46. An immobilized carbohydrate derivativebiosensor, comprising: a surface, a binding group, a carbohydratederivative which is capable of binding to at least one member selectedfrom the group consisting of a protein, a virus and a cell, wherein thecarbohydrate derivative, contains at least one O-, N-, C- orS-glycosidically bound aglycon, in which the aglycon contains at leastone aliphatic or aromatic compound, wherein the carbohydrate derivativeis bound to a spacer, wherein the aliphatic or aromatic compoundconsists of a spacer and the binding group, and the binding groupcomprises a member selected from the group consisting of —S, —NH—CO—,—CO—NH—, —NH—, and —N═N—, a protein is linked between the binding groupand the surface, and the protein is bound to the surface.
 47. Thebiosensor according to claim 46, wherein the protein comprises bovineserum albumin.